Structure and Engineering of Natural Product Cytochrome P450 Enzymes

描述（由申请人提供）：细胞色素p450是自然界中分布最广泛的酶类之一，催化广泛的天然产物和外源小分子的氧化。尽管已有数百种P450羟化酶在异种生物和类固醇的氧化代谢中被研究，但在细菌次生代谢中，特别是在大环内酯类抗生素的生物合成途径中，只有很少的P450羟化酶被研究。在大多数这些途径中，羟基化发生在聚酮合成酶（PKS）形成大环内酯后的生物合成后期。除了显著提高生物效力外，羟基化还为化学修饰和进一步增强抗感染活性提供了潜在的位点。因此，通过组合生物合成和化学酶合成创造新的大环内酯类似物需要同时努力开发具有广泛底物特异性的大环内酯单加氧酶。提出的工作的目的是全面了解细胞色素P450-PikC大环内酯单加氧酶的底物灵活性和功能。我们最近对该酶的结构测定为其催化机制和通过12元环大环内酯YC-17和14元环大环内酯narbomycin羟基化产生多种产物的能力提供了令人着迷的新见解。这些信息将指导蛋白质工程工作，以更好地了解酶的功能和位置特异性，以及它催化羟基化或环氧化反应的能力。此外，我们计划研究前所未有的去糖胺糖介导的大环内酯在PikC结合域内的锚定，以开发具有多种底物选择性的工程单加氧酶。具体目标PikC结构及纳博霉素与YC-17羟基化作用机制的测定。具体目标2。探讨大环内酯糖介导的锚定在PikC的底物结合和催化活性中的作用。具体目标3。新型大环内酯羟化酶的工程研究。

DESCRIPTION (provided by applicant): Cytochrome P450s are one of the most widely distributed classes of enzymes in nature, catalyzing the oxidation of a broad range of natural product and xenobiotic small molecules. Although hundreds of P450 hydroxylases have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these pathways, hydroxylation(s) occurs in the late stages of biosynthesis after formation of the macrolide by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of anti-infective activity. Thus, the creation of novel macrolide analogs through combinatorial biosynthesis and chemoenzymatic synthesis warrants a concomitant effort towards the development of macrolide monooxygenases with broad substrate specificity. The aim of the proposed work is to develop a thorough understanding of the substrate flexibility and functionality of the cytochrome P450-PikC macrolide monooxygenase. Our recent structure determination of the enzyme has provided fascinating new insights into its catalytic mechanism and ability to generate several products by hydroxylation of the 12-membered ring macrolide YC-17 and the 14-membered ring macrolide narbomycin. This information will direct protein engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze hydroxylation or epoxidation reactions. Moreover, we plan to investigate the unprecedented desosamine sugar-mediated anchoring of macrolides within the PikC binding domain to develop engineered monooxygenases with versatile substrate selectivity. Specific Aim 1. Determination of the PikC structure and the mechanism(s) of hydroxylation of narbomycin and YC-17. Specific Aim 2. Explore the role of macrolide sugar-mediated anchoring on substrate binding and catalytic activity of PikC. Specific Aim 3. Engineering of novel macrolide hydroxylases.